Chimeric Anti-Staphylococcal Enterotoxin B Antibodies and Lovastatin Act Synergistically to Provide In Vivo Protection against Lethal Doses of SEB

Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts

Interferon Gamma-Dependent Intestinal Pathology Contributes to the Lethality in Bacterial Superantigen-Induced Toxic Shock Syndrome

Toxic shock syndrome (TSS) caused by the superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes is characterized by robust T cell activation, profound elevation in systemic levels of multiple cytokines, including interferon-γ (IFN-γ), followed by multiple organ dysfunction and often death. As IFN-γ possesses pro- as well as anti-inflammatory properties, we delineated its role in the pathogenesis of TSS. Antibody-mediated in vivo neutralization of IFN-γ or targeted disruption of IFN-γ gene conferred significant protection from lethal TSS in HLA-DR3 transgenic mice. Following systemic high dose SEB challenge, whereas the HLA-DR3.IFN-γ+/+ mice became sick and succumbed to TSS, HLA-DR3.IFN-γ−/− mice appeared healthy and were significantly protected from SEB-induced lethality. SEB-induced systemic cytokine storm was significantly blunted in HLA-DR3.IFN-γ−/− transgenic mice. Serum concentrations of several cytokines (IL-4, IL-10, IL-12p40 and IL-17) and chemokines (KC, rantes, eotaxin and MCP-1) were significantly lower in HLA-DR3.IFN-γ−/− transgenic mice. However, SEB-induced T cell expansion in the spleens was unaffected and expansion of SEB-reactive TCR Vβ8+ CD4+ and CD8+ T cells was even more pronounced in HLA-DR3.IFN-γ−/− transgenic mice when compared to HLA-DR3.IFN-γ+/+ mice. A systematic histopathological examination of several vital organs revealed that both HLA-DR3.IFN-γ+/+ and HLA-DR3.IFN-γ−/− transgenic mice displayed comparable severe inflammatory changes in lungs, and liver during TSS. Remarkably, whereas the small intestines from HLA-DR3.IFN-γ+/+ transgenic mice displayed significant pathological changes during TSS, the architecture of small intestines in HLA-DR3.IFN-γ−/− transgenic mice was preserved. In concordance with these histopathological changes, the gut permeability to macromolecules was dramatically increased in HLA-DR3.IFN-γ+/+ but not HLA-DR3.IFN-γ−/− mice during TSS. Overall, IFN-γ seemed to play a lethal role in the immunopathogenesis of TSS by inflicting fatal small bowel pathology. Our study thus identifies the important role for IFN-γ in TSS

Epidermal Growth Factor Receptor Inhibition Is Protective in Hyperoxia-Induced Lung Injury.

AIMS: Studies have linked severe hyperoxia, or prolonged exposure to very high oxygen levels, with worse clinical outcomes. This study investigated the role of epidermal growth factor receptor (EGFR) in hyperoxia-induced lung injury at very high oxygen levels (\u3e95%). RESULTS: Effects of severe hyperoxia (100% oxygen) were studied in mice with genetically inhibited EGFR and wild-type littermates. Despite the established role of EGFR in lung repair, EGFR inhibition led to improved survival and reduced acute lung injury, which prompted an investigation into this protective mechanism. Endothelial EGFR genetic knockout did not confer protection. EGFR inhibition led to decreased levels of cleaved caspase-3 and poly (ADP-ribosyl) polymerase (PARP) and decreased terminal dUTP nick end labeling- (TUNEL-) positive staining in alveolar epithelial cells and reduced ERK activation, which suggested reduced apoptosis CONCLUSION: In conditions of severe hyperoxia (\u3e95% for \u3e24 h), EGFR inhibition led to improved survival, decreased lung injury, and reduced cell death. These findings further elucidate the complex role of EGFR in acute lung injury

In Vitro and In Vivo Evaluation of Staphylococcal Superantigen Peptide Antagonists

Superantigen peptide antagonists failed to block T-cell activation and cytokine production as well as toxic shock induced by staphylococcal enterotoxin B (SEB) in HLA class II transgenic mice. They also failed to inhibit the binding of SEB to HLA class II molecules as well as activation of human T lymphocytes in vitro

A randomised comparative evaluation of supraclavicular and infraclavicular approaches to brachial plexus block for upper limb surgeries using both ultrasound and nerve stimulator

Background and Aims: The supraclavicular and infraclavicular brachial plexus blocks have a similar distribution of anaesthesia, and both can be used effectively for surgeries of the upper limb. This study aimed to compare the supraclavicular and infraclavicular approaches of brachial plexus blocks, guided by ultrasound and neurostimulation. Methods: Sixty adult patients scheduled for elective upper limb surgery of the elbow and/or below were randomly divided into two groups: infraclavicular Group (I) and supraclavicular Group (S). All the blocks were performed with the aid of ultrasound-guided nerve stimulator confirmation. The two groups were compared with respect to block performance time, onset of sensory and motor blockade, readiness for surgery, success rate and complications. The statistical analysis was performed with Student t-test and Chi-square test. Results: The block performance time for the infraclavicular group was 9.57 ± 3.19 min, whereas for supraclavicular group, it was 11.53 ± 2.90 min with similar success rates (93.3%). Onset of sensory blockade was achieved earlier (6.43 ± 2.61 min) in Group I than Group S (8.45 ± 2.87 min, P = 0.006). The onset of motor blockade was similar in Group I (7.32 ± 2.90 min) and Group S (8.68 ± 3.50 min, P = 0.121). The patient satisfaction was similar in both the groups. One patient had a pneumothorax, three patients developed Horner syndrome and another had clinically symptomatic diaphragmatic paresis in Group S. Conclusion: The infraclavicular block is more rapidly executed compared to supraclavicular block with similar success rates and fewer complications in the presence of ultrasound and nerve stimulator and hence should be preferred

Early gene expression changes induced by the bacterial superantigen staphylococcal enterotoxin B and its modulation by a proteasome inhibitor

Toxic shock syndrome (TSS) is an acute, serious systemic illness caused by bacterial superantigens. Nonavailability of a suitable animal model until recently has hampered an in-depth understanding of the pathogenesis of TSS. In the current study, we characterized the early molecular events underlying TSS using our HLA-DR3 transgenic mouse model. Gene expression profiling using DNA microarrays identified a rapid and significant upregulation of several pro- as well as anti-inflammatory mediators, many of which have never been previously described in TSS. In vivo administration of staphylococcal enterotoxin B (SEB) led to an increase in the expression of Th0- (IL-2, 240-fold); Th1- (IFN-γ, 360-fold; IL-12, 8-fold); Th2- (IL-4, 53-fold; IL-5, 4-fold) as well as Th17-type cytokines (IL-21, 19-fold; IL-17, 5-fold). The immunoregulatory cytokines (IL-6, 700-fold; IL-10, 18-fold); CC chemokines (such as CCL 2, 11, 3, 24, 17, 12, 7), CXC chemokines (such as CXCL 1, 2, 5, 11, 10, 19); and several proteases (matrix metalloproteinases 13, 8, 3, and 9) were also upregulated. Serum levels of several of these cytokines/chemokines were also significantly elevated. Pathway analyses revealed significant modulation in a variety of biochemical and cellular functions, providing molecular insights into the pathogenesis of TSS. Administration of bortezomib, a clinically approved proteasome inhibitor capable of blocking NF-κB pathway, was able to significantly modulate the expression of a variety of genes induced by SEB. Thus, our study showed that TSS is a complex process and emphasized the potential of use of bortezomib in the therapy of superantigen-induced TSS